Compositions and methods for the targeted insertion of a nucleotide sequence of interest into the genome of a plant

Abstract

Methods for the targeted integration of nucleotide sequences into a plant are provided. Transfer cassettes comprising nucleotide sequences of interest flanked by non-identical recombination sites are used to transform a plant comprising a target site. The target site contains at least a set of non-identical recombination sites corresponding to those on the transfer cassette. Exchange of the nucleotide sequences flanked by the recombination sites is effected by a recombinase.

That which is claimed: 
     
       1. A method for targeting the insertion of a polynucleotide of interest to a specific chromosomal site in a genome of a plant cell, said method comprising:
 a) introducing into the plant cell a transfer cassette by particle bombardment, said transfer cassette comprising said polynucleotide of interest flanked by a first and a second recombination site, wherein said first and said second recombination sites are non-identical; and wherein said genome of said plant cell comprises a target site comprising the first and the second non-identical recombination sites of said transfer cassette; and 
 b) providing a recombinase that recognizes and implements recombination at the first and the second recombination sites wherein, the polynucleotide of interest is inserted into the genome of said plant cell at said target site. 
 
     
     
       2. The method of  claim 1 , wherein providing said recombinase comprises genetically transforming said plant cell with an expression cassette comprising a polynucleotide encoding said recombinase. 
     
     
       3. The method of  claim 1 , wherein said recombinase is FLP. 
     
     
       4. The method of  claim 3 , wherein said FLP has been synthesized using maize preferred codons. 
     
     
       5. The method of  claim 1 , wherein said recombination site comprises SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. 
     
     
       6. The method of  claim 1 , wherein said recombinase is Cre. 
     
     
       7. The method of  claim 6 , wherein said Cre recombinase has been synthesized using maize preferred codons. 
     
     
       8. The method of  claim 1 , wherein said plant cell is from a monocot. 
     
     
       9. The method of  claim 8 , wherein said monocot is maize. 
     
     
       10. The method of  claim 1 , wherein said plant cell is from a dicot. 
     
     
       11. The method of  claim 10 , wherein said dicot is canola,  Brassica , soybean, sunflower, or cotton. 
     
     
       12. The method of  claim 1 , wherein the genome of the plant cell further comprises an expression cassette comprising a polynucleotide encoding said recombinase wherein said recombinase is FLP. 
     
     
       13. The method of  claim 12 , wherein said recombinase is FLP and has been synthesized using maize preferred codons. 
     
     
       14. The method of  claim 1 , wherein said plant cell is in a plant. 
     
     
       15. The method of  claim 14 , wherein said plant is a monocot. 
     
     
       16. The method of  claim 15 , wherein said monocot is maize. 
     
     
       17. The method of  claim 14 , wherein said plant is a dicot. 
     
     
       18. The method of  claim 1 , wherein said first recombination site of the transfer cassette is in an intron. 
     
     
       19. The method of  claim 1 , wherein said first recombination site of the transfer cassette is in a 5′ UTR.